Abstract: The broader, long-term objectives of this proposal are to dissect the mechanisms governing alloimmune responses of adoptively transferred donor T cells after their administration to established allogeneic chimeras and to use this data to develop clinically translatable strategies to augment anti- tumor immunity. Our preliminary studies suggest that after MHC-matched allografting, host-derived dendritic cells (DCs) persist in the skin, despite the apparent full donor DC chimerism in the blood and ongoing graft-versus-leukemia (GVL) responses. Furthermore, we found that adoptive transfer of donor T cells to allogeneic MHC-matched chimeras, in which all blood DCs are of donor origin, can lead to graft-versus-host (GVH) reactivity toward residual host skin DCs. Interestingly, DLI administration to chimeras that have been topically treated with the Toll-like receptor (TLR)7 ligand, imiquimod, not only augmented the DLI-mediated GVH reactivity, but also the GVL response. These responses, if combined with tumor vaccination, resulted in the ability of treated animals to exhibit an anamnestic response to tumor re-challenge. Accordingly, the central hypothesis of this proposal is that donor T cell?host skin DC interactions play a principal role in the induction of an alloimmune response in complete donor chimeras post-transplant; and, moreover, that the outcome of these responses can be manipulated in vivo with defined molecular ligands and tumor vaccines to result in long-lasting anti-tumor immunity. To investigate this hypothesis, the following specific aims are proposed: 1) To characterize the cellular and molecular mechanisms governing T cell?DC interactions post MHC-matched allografting. These studies will determine the roles of host and donor DCs on the DLI-mediated GVH and GVL reactivities and characterize the role of TLR-mediated signaling; and 2) To determine the role of T cell?DC interactions on the vaccine-induced responses after allografting. These studies will examine the functional significance of residual host skin DCs and donor blood DCs on the induction of antigen-specific T cells and vaccine-induced immunity. The immediate goal of the studies described in this proposal is to develop novel therapeutic strategies that may lead to improved anti- tumor immunity; the long-term goal is to translate these findings to the clinic. Scope of the work: The central hypothesis behind this proposal is that interactions between donor T cells and residual host skin DCs play a principal role in the induction of alloimmune responses in complete donor chimeras post-transplant and that outcome of these responses can be manipulated in vivo with defined molecular ligands and tumor vaccine resulting in long-lasting anti-tumor immunity. Thus, the goal of the proposed study is to dissect the mechanisms governing alloimmune responses in established MHC- matched allogeneic chimeras and to use this data to develop clinically translatable strategies to augment anti-tumor immunity.